本文選題：單域抗體 + HER2��； 參考：《暨南大學(xué)》2016年博士論文

【摘要】：目的:HER2和FGFR2蛋白在多種癌細(xì)胞內(nèi)過(guò)表達(dá),與多種癌癥的發(fā)生、發(fā)展和轉(zhuǎn)移有關(guān),是癌癥治療的理想靶標(biāo)。雖然已有抗HER2的抗體藥上市用于癌癥治療,但這些都是傳統(tǒng)的單克隆抗體。由于單克隆抗體是大分子蛋白,其應(yīng)用受到很大限制。本論文篩選抗HER2的新型的小分子人源單域抗體,希望能代替上市的單克隆抗體藥物。另外,也篩選抗FGFR2的新型的小分子人源單域抗體。方法:第一部分,篩選抗HER2的人源單域抗體。采用輔助噬菌體侵染制備人源單域抗體噬菌體文庫(kù),采用原核細(xì)胞包涵體表達(dá)方法制備和純化HER2-domain II蛋白片段。采用噬菌體展示技術(shù)和HER2-domain II蛋白片段作為抗原篩選人源單域抗體文庫(kù)。經(jīng)過(guò)5輪文庫(kù)的篩選富集后,采用多克隆噬菌體ELISA和單克隆噬菌體ELISA方法進(jìn)一步篩選抗HER2-domain II的單域抗體,并獲得單域抗體的DNA序列。采用原核細(xì)胞可溶性表達(dá)方法制備和純化抗HER2-domain II的單域抗體蛋白,采用ELISA鑒定純化的單域抗體與HER2-domain II的結(jié)合能力,采用非競(jìng)爭(zhēng)性ELISA檢測(cè)單域抗體與HER2-domain II的親和力。第二部分,篩選抗FGFR2的人源單域抗體。采用輔助噬菌體侵染制備人源單域抗體噬菌體文庫(kù),通過(guò)人工合成的方法制備FGFR2蛋白片段。采用噬菌體展示技術(shù)篩選抗FGFR2蛋白片段的單域抗體,經(jīng)過(guò)5輪文庫(kù)的篩選富集后,采用多克隆噬菌體ELISA和單克隆噬菌體ELISA方法進(jìn)一步篩選抗FGFR2蛋白片段的單域抗體,并獲得單域抗體的DNA序列。結(jié)果:第一部分,篩選抗HER2的人源單域抗體。利用輔助噬菌體侵染制備了滴度為3.41×1013/ml的人源單域抗體噬菌體文庫(kù)。通過(guò)原核細(xì)胞包涵體表達(dá)純化得到了HER2-domain II蛋白,SDS-PAGE電泳圖顯示較高的蛋白純度;非還原性SDS-PAGE電泳圖顯示HER2-domain II蛋白約為12 kDa。經(jīng)過(guò)噬菌體展示技術(shù)5輪篩選后,抗HER2-domain II的單域抗體的滴度逐漸上升。采用多克隆噬菌體ELISA檢測(cè)發(fā)現(xiàn)第3輪富集的單域抗體具有最強(qiáng)的的結(jié)合能力。從篩選后的文庫(kù)里隨機(jī)挑選196個(gè)單域抗體克隆,采用單克隆噬菌體ELISA檢測(cè)得到了8個(gè)陽(yáng)性單域抗體;采用單克隆噬菌體ELISA和多個(gè)無(wú)關(guān)抗原檢測(cè)發(fā)現(xiàn)這8個(gè)陽(yáng)性單域抗體能與HER2-domain II特異性結(jié)合。對(duì)于這8個(gè)陽(yáng)性單域抗體的DNA測(cè)序發(fā)現(xiàn)只有2個(gè)不同的單域抗體(分別命名為aHER2-1C1和aHER2-2B4)。將這2個(gè)單域抗體克隆到pET22b表達(dá)載體內(nèi)進(jìn)行可溶性表達(dá)純化,SDS-PAGE電泳圖顯示得到了較高純度的蛋白。ELISA檢測(cè)發(fā)現(xiàn)純化的a HER2-1C1能與HER2-domain II結(jié)合,而純化的aHER2-2B4與HER2-domain II結(jié)合的能力弱。采用非競(jìng)爭(zhēng)性ELISA檢測(cè)發(fā)現(xiàn)aHER2-1C1與HER2-domain II結(jié)合的親和力為3.76 uM。第二部分,篩選抗FGFR2的人源單域抗體。通過(guò)人工合成的方法得到了FGFR2蛋白片段,高效液相色譜顯示較高的蛋白純度,質(zhì)譜分析顯示FGFR2蛋白片段約為4 kDa。經(jīng)過(guò)噬菌體展示技術(shù)對(duì)人源單域抗體噬菌體文庫(kù)進(jìn)行了5輪篩選,采用多克隆噬菌體ELISA檢測(cè)發(fā)現(xiàn)第5輪富集的單域抗體具有最強(qiáng)的的結(jié)合能力。從篩選后的文庫(kù)里隨機(jī)挑選168個(gè)單域抗體克隆,采用單克隆噬菌體ELISA檢測(cè)得到了20個(gè)陽(yáng)性單域抗體;采用單克隆噬菌體ELISA和多個(gè)無(wú)關(guān)抗原檢測(cè)發(fā)現(xiàn)這20個(gè)陽(yáng)性單域抗體能與FGFR2蛋白片段特異性結(jié)合。對(duì)于這20個(gè)陽(yáng)性單域抗體的DNA測(cè)序發(fā)現(xiàn)只有2個(gè)不同的單域抗體(分別命名為aFG-7E5和aFG-9F8)。結(jié)論:本研究采用噬菌體展示技術(shù)分別獲得了與HER2-domain II和FGFR2蛋白片段特異性結(jié)合的人源單域抗體,為癌癥的早期診斷與靶向治療奠定了較好的基礎(chǔ)。
[Abstract]:Objective: HER2 and FGFR2 protein are overexpressed in a variety of cancer cells, which are related to the occurrence, development and metastasis of various cancers. It is an ideal target for cancer treatment. Although anti HER2 antibody drugs have been used for cancer treatment, these are traditional monoclonal antibodies. The application of monoclonal antibodies is large, and its application is greatly limited. In this paper, a new type of small molecular human single domain antibody against HER2 is screened in the hope of replacing the listed monoclonal antibody drugs. In addition, a new type of small molecular human single domain antibody against FGFR2 is screened. Method: the first part is to screen the human single domain antibody against HER2. HER2-domain II protein fragment was prepared and purified by the expression of prokaryotic cell inclusion body. The phage display technique and HER2-domain II protein fragment were used as antigen to screen the single domain antibody library of human source. After screening and enrichment of 5 rounds of library, polyclonal phage ELISA and monoclonal phage ELISA were used to further screen the anti HER2-dom. The single domain antibody of AIN II and the DNA sequence of the single domain antibody were obtained. The single domain antibody protein of anti HER2-domain II was prepared and purified by the prokaryotic expression method, and the binding ability of the purified single domain antibody to HER2-domain II was identified by ELISA, and the affinity of the non competitive ELISA detection single domain antibody to HER2-domain II was used. Second Part, screening anti FGFR2 human single domain antibody. Using phage infection to prepare human single domain antibody phage library, FGFR2 protein fragment was prepared by artificial synthesis method. Single domain antibody of anti FGFR2 protein fragment was screened by phage display technique. After screening and enrichment of 5 rounds of library, polyclonal phage ELISA was used. The monoclonal phage ELISA method further screened the single domain antibody against the FGFR2 protein fragment and obtained the DNA sequence of the single domain antibody. Results: the first part was to screen the human single domain antibody against HER2. The phage library of human single domain antibody of 3.41 x 1013/ml was prepared by the auxiliary phage infection. The expression of the inclusion body of the prokaryotic cell was expressed. The purified HER2-domain II protein was obtained, and the SDS-PAGE electrophoresis map showed high protein purity, and the non reductive SDS-PAGE electrophoresis map showed that the HER2-domain II protein was about 12 kDa. after 5 rounds of phage display technology, the titer of the single domain antibody against HER2-domain II increased gradually. The polyclonal phage ELISA detection found third rounds of enrichment. The single domain antibody had the strongest binding ability. 196 single domain antibody clones were randomly selected from the screened library and 8 positive single domain antibodies were detected by the monoclonal phage ELISA detection. The 8 positive single domain antibodies were found to be specific to HER2-domain II by monoclonal phage ELISA and multiple independent antigens. DNA sequencing of these 8 positive single domain antibodies found only 2 different single domain antibodies (named aHER2-1C1 and aHER2-2B4 respectively). The 2 single domain antibodies were cloned into the pET22b expression vector for soluble expression and purification. The SDS-PAGE electrophoresis map showed that the purified egg white.ELISA detection was found to be the purified a HER2-1C1 and HER2. -domain II was combined, and the ability of the purified aHER2-2B4 to bind to HER2-domain II was weak. The affinity of aHER2-1C1 to HER2-domain II was found to be 3.76 uM. second by non competitive ELISA detection, and the anti FGFR2 human single domain antibody was screened. The FGFR2 protein fragment was obtained by artificial synthesis, and high performance liquid chromatography showed higher performance. The protein purity and mass spectrometry analysis showed that the FGFR2 protein fragment was about 4 kDa., and the phage display technique was used to screen the human single domain antibody phage library by 5 rounds. The polyclonal phage ELISA detection showed that the fifth round of single domain antibodies had the strongest binding ability. 168 single domains were randomly selected from the selected library. 20 positive single domain antibodies were obtained by monoclonal phage ELISA detection. The 20 positive single domain antibodies were identified by monoclonal phage ELISA and multiple independent antigens. The DNA sequencing of the 20 positive single domain antibodies found only 2 different single domain antibodies (named respectively. AFG-7E5 and aFG-9F8) conclusion: This study uses phage display technique to obtain a human single domain antibody with specific binding of HER2-domain II and FGFR2 protein fragments, which lays a good foundation for early diagnosis and targeting therapy of cancer.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R730.51

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